Method for inactivating enzymes, microorganisms and spores in a liquid foodstuff

ABSTRACT

A method for (1) inactivating enzymes, microorganisms and spores in a liquid foodstuff, and (2) sterilizing microorganisms, spores and virus in a liquid foodstuff, comprises contacting the liquid foodstuff to be treated with carbon dioxide of a supercritical state supplied through a filter with a mesh size of not more than 120 μm in average diameter (average size) in a treating tank.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of United States patent application, U.S. Ser. No. 08/291,484 filed on Aug. 17, 1994, now U.S. Pat. No. 5,520,943.

BACKGROUND OF THE INVENTION

The present invention relates to a method for (1) inactivating enzymes, microorganisms and spores in a liquid foodstuff, and (2) sterilizing microorganisms, spores and virus in a liquid foodstuff. More particularly, the present invention relates to a method for (1) inactivating enzymes, microorganisms and spores in a liquid foodstuff, and (2) sterilizing microorganisms, spores and virus in a liquid foodstuff, which comprises contacting the liquid foodstuff with carbon dioxide in a supercritical state supplied through a filter, so that this method is highly safe in practice thereof and, further does not require specific high-temperature heating step or heating step under reduced pressure.

Soy sauce which is one of fermented foodstuffs, is a typical Japanese seasoning which has gained popularity overseas along with dishes of Japanese cuisine in recent years. Soy sauce, however, is disliked in part by Japanese and foreigners because of its peculiar smell. It is, therefore, considered a key factor for elevating the utility and value of soy sauce to remove the peculiar smell (odor) of soy sauce and to enhance its mild aroma.

Sake, which is another fermented foodstuff, is generally produced through the following steps. In the first step, fermented rice is compressed and filtered to obtain shinshu (green sake). In the second step, this obtained green sake is sterilized by heating at 60° to 65° C. and stored. In the third step, a number of lots of the stored sake which have been heat-treated are properly blended to determine the sake quality (sweet or dry, etc.) and the alcohol content is adjusted to standards. In the fourth (final) step, the thus prepared sake is again sterilized by heating and then bottled or packed. Thus, in the production of sake the heat-treatment is conducted twice, in the second and forth steps, in the manufacturing process, to effect inactivation of enzymes and sterilization.

The fresh flavor of green sake is sharply reduced by two heat-treatments in the manufacturing process, so that consumers request fresh sake which has been produced without undergoing any heat-treatment. However, green sake, although fresh in taste and smell, is very changeable in flavor because of its high enzymatic activity. There are marketed some brands of non-heat-treated sake sold at low temperature as fresh sake, but such fresh sake is subject to deterioration of quality by the action of certain enzymes such as α-amylase, protease and carboxy-peptidase, which poses the problem of increased distribution costs to prevent qualitative deterioration.

In order to solve the above problem, attempts have been made to remove such enzymes in the green sake by precision filtration or ultra-filtration, but sufficient removal of the enzymes has been impossible with such means. Further, the enzyme removal using a membrane has the problem that the aromatic components of green sake may be dissolved in the membrane during the process and/or that some kinds of taste components of green sake may be removed, thereby losing its relish.

Also, with reference to muddled fruit drinks such as orange juice, etc., it is considered that the cloud of the drink is a decisive factor for the quality of the drink. Inactivation of pectin esterase (PE) is essential for maintaining the stability of this cloud, but since PE is an enzyme stable to heat, usually a heat-treatment under a high-temperature condition (88° to 99° C. or 120° C.) is required for inactivation of this enzyme. However, the heat-treatment at such high temperature causes deterioration of freshness and characterstic taste of the drink.

On the other hand, Japanese Patent Application Laid-Open (KOKAI) No. 3-27268 discloses a method for producing soy sauce, comprising bringing soy sauce into contact with carbon dioxide kept in liquid state or supercritical state to obtain soy sauce reduced in smell characteristic thereof and having a good disappearance of foam.

U.S. Pat. No. 4,714,591 discloses a method to recover valuable lignin and other extractable components from kraft black liquor and remove undesired components therefrom using supercritical fluids and convert the recovered lignin into chemical products. Further, U.S. Pat. No. 4,714,591 discloses a method for inactivating enzymes, comprising passing supercritical carbon dioxide through orange juice. JOURNAL OF FOOD SCINCE, Vol. 56, No. 3, 743-746, 1991 discloses a method for inactivating enzymes, comprising passing supercritical carbon dioxide through orange juice. U.S. Pat. No. 3,966,981 discloses a process for removing residual solvent from materials containing the same which involves extracting the material with liquid CO₂, separating the phases, and evaporating CO₂ from the treated material. Agric. Biol. Chem., 51 (2), 407-412, 1987 discloses a method for sterilizing microorganisms with supercritical carbon dioxide. Hakkokogaku, Vol. 67, No. 5, 439-457, 1989 disclosures a supercritical carbon dioxide extraction method.

However, there is no teaching nor suggestion of supplying carbon dioxide of a supercritical state as a micro-particle fluid state into a reactor by means of a filter in Japanese Patent Application Laid-Open (KOKAI) No. 3-27268, U.S. Pat. No. 4,714,591, JOURNAL OF FOOD SCINCE, Vol. 56, No. 3, 743-746, 1991, U.S. Pat. No. 3,966,981, Agric. Biol. Chem., 51 (2), 407-412, 1987 and Hakkokogaku, Vol. 67, No. 5, 439-457, 1989. By the methods disclosed in the above-mentioned publications, it is impossible to obtain sufficient amelioration of smell, inactivation of enzymes and sterilization. Further, it is impossible from the above-mentioned publications to expect the supplying carbon dioxide of a supercritical state as a micro-particle fluid state through a filter with a specific mesh size for (1) inactivating enzymes, microorganisms and spores in a liquid foodstuff, and (2) sterilizing microorganisms and virus in a liquid foodstuff.

As a result of intensive studies for solving the above problems, it has been found that by contacting a liquid foodstuff such as fermented liquid foodstuffs and various kinds of fruit juice with carbon dioxide of a supercritical state as a micro-particle fluid state, supplied through a filter with a mesh size of not more than 120 μm in average diameter in a treating tank, the desired inactivation of enzymes, microorganisms and spores, and sterilization of microorganisms, spores and virus can be accomplished effectively. On the basis of the finding, the present invention has been attained.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a method for inactivation of enzymes, microorganisms and spores in a liquid foodstuff such as fermented liquid foodstuffs and various kinds of fruit juice, which is high safety in practice and avoids high temperature heat-treatment.

Another object of the present invention is to provide a method for the sterilizing microorganisms, spores and virus in a liquid foodstuff such as fermented liquid foodstuffs and various kinds of fruit juice, which is high safety in practice and avoid high temperature heat-treatment.

To attain the aims, in a first aspect of the present invention, there is provided a method for inactivating enzymes, microorganisms and spores in a liquid foodstuff such as fermented liquid foodstuffs and various kinds of fruit juice, comprising contacting the liquid foodstuff to be treated with carbon dioxide of a supercritical state supplied through a filter with a mesh size of not more than 120 μm in average diameter (average size) in a treating tank.

In a second aspect of the present invention, there is provided a method for sterilizing microorganisms, spores and phage virus in a liquid foodstuff such as fermented liquid foodstuffs and various kinds of fruit juice, comprising contacting the liquid foodstuff to be treated with carbon dioxide of a supercritical state supplied through a filter with a mesh size of not more than 120 μm in average diameter (average size) in a treating tank.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a block diagram illustrating a treating method of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The liquid foodstuffs to which the method of the present invention can be applied include, as typical examples, fermented liquid foodstuffs such as sake, beer, wine and the like, and various kinds of fruit juice. The fruit juices contemplated as "liquid foodstuff" in the present invention are those prepared from apple, grape and various kinds of citrus fruits, but they also include the juices produced from tomato and other vegetables.

In the present invention, carbon dioxide is used as a supercritical fluid obtained by pressurizing carbon dioxide under a pressure of 70 to 400 atm, preferably 100 to 300 atm, more preferably 150 to 300 atm, and the temperature is 30° to 70° C., preferably 30° to 50° C. Carbon dioxide in its supercritical state may hereinafter be referred to as "carbon dioxide fluid".

It is required in the present invention that the carbon dioxide fluid be brought into sufficient contact with the liquid foodstuff to be treated. For attaining this, in the present invention, the carbon dioxide fluid is supplied as a micron fluid state (micro-particle fluid state) into the liquid foodstuff. More specifically, carbon dioxide of a supercritical state is supplied through a filter with a mesh size of not more than 120 μm in average diameter and brought into contact with the liquid foodstuff in a treating tank. Namely, the upper limit of the mesh size (average diameter) of the filter is 120 μm, preferably 100 μm, more preferably 50 μm, still more preferably 30 μm. The lower limit of the mesh size (average diameter) of the filter is 1 μm, preferably 2 μm. The kind of the filter used in the present invention is not specified, but it is recommended to use a filter made of a sintered metal such as copper, bronze, stainless steel, nickel, monel metal, silver, platinum or the like.

The pressure at contacting between the liquid foodstuff and the carbon dioxide fluid in the treating tank is variable depending on the kind of the liquid foodstuff to be treated and the desired degree of modification. For example, the lower limit of the pressure is 7 MPa, preferably 10 MPa, more preferably 15 MPa, still more preferably 20 MPa. The upper limit of the pressure is 50 MPa, preferably 30 MPa.

The temperature at contacting between the liquid foodstuff and the carbon dioxide-fluid in the treating tank is variable depending on the kind of the liquid foodstuff to be treated and the desired degree of modification. For example, the lower limit of the temperature is 30° C., preferably 35° C. The upper limit of the temperature is 80° C., preferably 50° C.

The contact time between the liquid foodstuff and the carbon dioxide fluid in the treating tank is variable depending on the kind of the liquid foodstuff to be treated and the desired degree of modification. For example, the upper limit of the contact time is 120 minutes, preferably 60 minutes, more preferably 30 minutes. The lower limit of the contact time is 10 seconds, preferably 20 seconds, more preferably 30 seconds.

The lower limit of the carbon dioxide (CO₂) density in the liquid foodstuff in the treating tank is 0.4 g/cm³, preferably 0.6 g/cm³, more preferably 0.72 g/cm³, still more preferably 0.85 g/cm³. The lower limit of the carbon dioxide (CO₂) concentration in the liquid foodstuff in the treating tank is 0.4 mole/liter, preferably 0.6 mol/liter, more preferably 0.7 mol/liter.

In the present invention, the addition of an entrainer does usually not required, but if necessary, it may be added in an amount of not more than 5 wt % based on the carbon dioxide fluid. In the case of the liquid foodstuff containing no alcoholic substance, ethyl alcohol may be used as entrainer. The treatment with the carbon dioxide fluid may be either batchwise or continuous.

The liquid foodstuff which has been subjected to contacting-treatment with the carbon dioxide fluid can be easily recovered by restoring the ordinary temperature and ordinary pressure conditions to the liquid foodstuff. The recovered liquid foodstuff remains unaffected in flavor, even though enzymes, microorganism and spores therein have been inactivated or microorganisms, spores and virus therein have been sterilized. Also, the foodstuff treated by the carbon dioxide fluid is not deteriorated in quality during storage.

As the microorganisms contained in the liquid foodstuff, microorganisms belonging to genus Lactobacillus, genus Streptcoccus, genus Pseudomonas, genus Bacillus, genus Clostridium, genus Acetobacter, genus Saccharomyces may be exemplified. Further, as the species of the microorganisms, Lactobacillus brevis, Lactobacillus homohiochi, Lactobacillus bulgaricus, Bacillus coil, Bacillus cereus, Bacillus subtilis, Bacillus megaterium, Bacillus polymyxa, Bacillus coagulans, Bacillus spores, Penicillium frequentans, Penicillium jenseni, Clostridium thyrobutyricum, Acetobacter aceti, Acetobacter suboxydans, Aspergillus oryzae, Saccharomyces pasteurianus and Saccharomyces cerevisiae may be exemplified.

As the enzymes contained in the liquid foodstuff, Protease, β-glucanase, Invertase, Amylase (α-Amylase, β-Amylase), Glucoamylase, Pectinase, Naringinase, Hesperidinase, Anthocyanase, Carboxypeptidase, Lipase, Ascorbic oxidase, Acid protease, Catalase, Lipoxigenase, Alkaline protease and Polyphenol oxidase may be exemplified.

As the spores, spores of Bacillus and Clostridium may be cited, and spores of Bacillus cereus, Bacillus subtilis, Bacillus megaterium, Bacillus polymyxa, Bacillus coagulans, Bacillus circulans, Bacillus licheniformis, Bacillus macerans and spore of Clostridium thyrobutyricum may be exemplified.

As the phage virus, Escherichia coli T1 phage, Escherichia coli T2 phage, Escherichia coli T3 phage, Escherichia coli T4 phage and Escherichia coli T5 phage may be exemplified.

In the process shown in FIG. 1, an entrainer container 2 is provided as occasion demands, and a treating tank 13 includes a pressure vessel. The internal temperature and pressure of the treating tank 13 can be adjusted to maintain a constant by a tank heater 14 and a pressure regulator 15. A filter 16 is provided at the bottom of the treating tank 13. The internal temperature and pressure of a separating tank 17 can be adjusted by a tank heater 18 and a pressure regulator 19.

In operation, carbon dioxide is led out from a liquefied carbon dioxide gas cylinder 1, passes through a valve 9 and a filter 5, and (after cooling carbon dioxide by a cooler 6 so as to liquefy carbon dioxide, when carbon dioxide is in a gaseous form) is supplied into a line mixer 8 through a heater 7 and a check valve 10 by a pump 3. In case an entrainer is used, it is supplied from an entrainer container 2 into the line mixer 8 through a check valve 11 by a pump 4 and mixed with carbon dioxide in the line mixer 8.

Then carbon dioxide (and entrainer) is passed through a valve 12 and a filter 16 to enter the treating tank 13, in which the liquid foodstuff to be treated is contained. Carbon dioxide is turned into a supercritical state by properly operating the pump 3, the heater 7, the tank heater 14 and the pressure regulator 15, and supplied in this supercritical state into the treating tank 13. Also, carbon dioxide is supplied in micron size (micro-particle) by passing through the filter 16 into the treating tank 13. It is also possible to provide carbon dioxide turned into a supercritical state as it is supplied into the treating tank 13.

The carbon dioxide fluid contacted with the liquid foodstuff is led into the separating tank 17 where the carbon dioxide fluid is released from its supercritical state by operating the tank heater 18 and the pressure regulator 19. The gasified carbon dioxide is discharged out of the system through a flowmeter 20. A valve 21 is provided as a preliminary means for discharging carbon dioxide out of the system at a halfway point before supplied into the treating tank 13.

The modification method according to the present invention has a high operational safety, requires no high-temperature heating and is capable of producing excellent effects in inactivation of enzymes, microorganisms and spores in a liquid foodstuff and excellent sterilization of microorganisms, spores and virus in a liquid foodstuff.

EXAMPLES

The present invention is further illustrated below with reference to the examples. These examples, however, are merely intended to be illustrative and not to be construed as limiting the scope of the invention in any way.

Test Example 1

A test liquid containing the substances having an offensive odor and a carbon dioxide fluid were contacted with each other according to the process illustrated in FIG. 1. As the test liquid, an aqueous solution containing hexanal, octanal, decanal, hexanol, decanol and undecanol, each in a concentration of 50 ppm, was used. A filter made of a sintered metal (SUS 316) and having a mesh size of 10 μm in average diameter was used as a filter 16 provided at the bottom of a treating tank 13. No entrainer was used.

First, 450 ml of the test liquid containing the offensive odor-substances was supplied into the treating tank 13 (500 ml) while starting the feed of carbon dioxide from a liquefied carbon dioxide gas cylinder 1. Carbon dioxide was passed through a valve 9 and a filter 5, was liquefied by a cooler 6, was heated to 40° C. by a heater 7 and was supplied into the treating tank 13 through a check valve 10, a line mixer 8, a valve 12 and the filter 16 by a pump 3. The pressure and temperature in the treating tank 13 were maintained at 200 atm and 40° C., respectively, by a tank heater 14 and a pressure regulator 15. The feeding rate of the carbon dioxide fluid into the treating tank 13 was set at 10 NL/min, and the contact time between the offensive odor substances-containing test liquid and the carbon dioxide fluid was predetermined to be 40 minutes.

Then, the offensive odor substances-containing test liquid and the carbon dioxide fluid were led into a separating tank 17, and the temperature and pressure of the carbon dioxide fluid in the separating tank 17 were returned to the ordinary level by operating a tank heater 18 and a pressure regulator 19 to release the supercritical state. The gasified carbon dioxide was discharged out of the system through a flowmeter 20. Gas chromatographic analysis of the offensive odor substances-containing test liquid after treatment according to the present invention showed that not less than 95 wt % of each of the offensive odor substances had been removed.

Example 1

450 ml of commercial soy sauce using soybean as a raw material was used as a liquid foodstuff to be treated, and it was contacted with a carbon dioxide fluid of micron size under the conditions of Test Example 1. Gas chromatographic analysis of the soy sauce after the treatment showed that more than 90 wt % of each of the volatile component substances in the soy sauce had been removed. Also, the result of an sensory test by the panelists on the treated soy sauce testified amelioration of mild aroma of the soy sauce after the treatment.

Volatile Component in Soy Sauce:

1,4-Diethylbenzene

Ethyl lactate

Furfural

2,5-Dihydro-2,5-dimethoxyfuran

Dihydroxy-5-methyl-2(3H)-furanone

Isovaleric acid

3-Methylthio-1-propanol

2-Acetylpyrrole

4-Hydroxy-2-ethyl-5-methyl-3(2H)-furanone

Test Example 2

An enzyme-containing test liquid and a carbon dioxide fluid were contacted with each other according to the process illustrated in FIG. 1. As the test liquid, an aqueous solution containing gluco amylase adjusted to a concentration of 20 mg/100 ml, was used. A filter made of a sintered metal (SUS 316) and having a mesh size of 10 μm in average diameter was used as a filter 16 provided at the bottom of a treating tank 13. No entrainer was used.

First, 450 ml of the test liquid was supplied into the treating tank 13 (500 ml) while starting the feed of carbon dioxide from a liquefied carbon dioxide gas cylinder 1. Carbon dioxide was passed through a valve 9 and a filter 5, was liquefied by a cooler 6, was heated to 35° C. by a heater 7 and was supplied into the treating tank 13 through a check valve 10, a line mixer 8, a valve 12 and the filter 16 by a pump 3. The pressure and temperature in the treating tank 13 were maintained at 250 atm and 35° C., respectively, by a tank heater 14 and a pressure regulator 15. The feeding rate of the carbon dioxide fluid into the treating tank 13 was 10 NL/min, and the contact time between enzyme-containing test liquid and the carbon dioxide fluid was 30 minutes.

Then, the enzyme-containing test liquid and the carbon dioxide fluid were led into a separating tank 17, and the temperature and pressure of the carbon dioxide fluid in the treating tank 17 were returned to the ordinary level by operating a tank heater 18 and a pressure regulator 19 to release the supercritical state. The gasified carbon dioxide was discharged out of the system through a flowmeter 20. In order to confirm the micron-size effect of the carbon dioxide fluid, the same test was conducted by removing the filter 16. As seen from the test results shown in Table 1, the residual enzyme activity could be markedly reduced by use of the filter.

                  TABLE 1                                                          ______________________________________                                         Condition    Filter was used                                                                           No filter was used                                     ______________________________________                                         Residual enzyme                                                                             17.3       46.4                                                   activity                                                                       ______________________________________                                    

Test Example 3

An aqueous solution containing carboxypeptidase (serine carboxypeptidase), lipase, glucoamylase and acidic protease, each in a concentration of 20-100 mg/100 ml, was used as an enzyme-containing test liquid. This enzyme-containing test liquid and a micron-size carbon dioxide fluid were contacted with each other following the same procedure as Test Example 2 except that the temperature of the carbon dioxide fluid supplied into the treating tank 13 and the pressure and temperature in the treating tank 13 were changed as shown in Tables 2 and 3. The ratio (%) of the residual enzyme activity in the solution used for treating the carbon dioxide fluid to the enzyme activity in the non-treated solution was determined, the results being shown in Tables 2 and 3.

                  TABLE 2                                                          ______________________________________                                                   Enzyme                                                                         Carboxypeptidase                                                                               Lipase                                                         Temp. (°C.)                                                   Pressure    35     40         35   40                                          ______________________________________                                         100 atm     14.13  0.00       0.17 0.00                                        150 atm     0.00   0.00       0.00 0.00                                        250 atm     0.00   0.00       0.00 0.00                                        ______________________________________                                    

                  TABLE 3                                                          ______________________________________                                                   Enzyme                                                                         Gluco amylase   Acidic protease                                                Temp. (°C.)                                                   Pressure    35     50         45   50                                          ______________________________________                                         100 atm     60.52  0.00       47.89                                                                               12.81                                       150 atm     59.45  0.00       35.26                                                                               11.23                                       250 atm     17.46  0.00       11.28                                                                               3.80                                        ______________________________________                                    

Example 2

450 ml of green sake was used as a liquid foodstuff, and it was contacted with a carbon dioxide fluid of micron size following the same procedure as Test Example 2. The residual enzyme activity (U/ml in sake) in the obtained sake after treatment with the carbon dioxide fluid and Lactobacillus brevis cell count (CFU/ml in sake) after 60-day storage are shown in Table 4. By way of comparison, the residual enzyme activity in the non-treated green sake (Comparative Example 1) and that in the heat-treated sake at 62° C. for 5 minutes (Comparative Example 2) are also shown in Table 4. The result of a sensory test by the panelists on the sake which had been treated with the carbon dioxide fluid (CO₂ -treated sake), showed that the CO₂ -treated sake had a good bouquet close to that of non-heated sake. On the other hand, in the result of the sensory test, the sake which had been subjected to the heat-treatment (Comparative Example 2) was inferior to non-heated sake in bouquet. The Lactobacillus brevis cell count was determined according to the calibration method using absorbance (at wavelength of 562 nm).

                  TABLE 4                                                          ______________________________________                                                             Comp.    Comp.                                                       Example 2 Example 1                                                                               Example 2                                         ______________________________________                                         Gluco amylase                                                                              0           330      0                                             Acidic protease                                                                            1.8         20.1     2.7                                           Carboxypeptidase                                                                           0           115      0                                             Lactobacillus                                                                              0           1.0 × 10.sup.9                                                                    0                                             brevis cell count                                                              ______________________________________                                    

In the above Example and Comparative Examples, the sake was kept in storage for 60 days and the passage with time of the concentrations of lactic acid and acetic acid were determined. The results are shown in Table 5. As is seen from Table 5, according to the enzyme inactivation process of the present invention, sterilization of the microorganisms such as lactic bacteria is simultaneously accomplished, thus preventing any elevation of concentration of lactic acid or acetic acid during the storage.

                  TABLE 5                                                          ______________________________________                                                              Comp.     Comp.                                                        Example 2                                                                              Example 1 Example 2                                       ______________________________________                                         Lactic After 20 days                                                                              365       360     355                                       acid   After 40 days                                                                              365       390     355                                       (ppm)  After 60 days                                                                              355       430     350                                       Acetic After 20 days                                                                               45        55      50                                       acid   After 40 days                                                                               45        65      50                                       (ppm)  After 60 days                                                                               45        70      50                                       ______________________________________                                    

Example 3

U.S.-grown navel oranges available on the market were squeezed by a hand presser to produce a juice, and 450 ml of this obtained juice was used as liquid foodstuff and contacted with a carbon dioxide fluid of micron size under the same conditions as Test Example 2. The pectin esterase (PE) activity in the juice after the treatment with the carbon dioxide fluid was determined, and the ratio (%) of the residual enzyme activity in this treated juice to the enzyme activity in the non-treated juice was calculated, the results being shown in Table 6. In Comparative Example 3, the same treatment and test were conducted by removing the filter 16.

Determination of the PE activity was made according to the Rouse Atkins method (1955). That is, a 1 wt % pectin solution was used as a substrate solution, and it was reacted with the enzyme at 30° C. for 30 minutes and the produced acid was titrated with a 0.02N sodium hydroxide solution to determine the enzymatic activity. An automatic titrator (manufactured by METROM CO., LTD.) was used for the titration.

                  TABLE 6                                                          ______________________________________                                                                Comp.                                                                  Example 3                                                                              Example 3                                               ______________________________________                                         Residual enzyme  0.1       39.5                                                activity (%)                                                                   ______________________________________                                    

Example 4

U.S.-grown Valencia oranges available on the market were squeezed by a hand presser to produce a juice and 450 ml of this obtained juice was used as a test liquid foodstuff. This juice and a carbon dioxide fluid of micron size were contacted with each other by following the same procedure as Test Example 2 except that the temperature of the carbon dioxide fluid supplied into the treating tank 13 and the temperature in the treating tank 13 were changed to 45° C. The residual PE activity (%) was calculated in the same way as Example 3. In Comparative Example 4, the same treatment and test were conducted by removing the filter 16. The results are shown in Table 7.

                  TABLE 7                                                          ______________________________________                                                               Comp.                                                                   Example 4                                                                             Example 4                                                ______________________________________                                         Residual enzyme  6.1      43.1                                                 activity (%)                                                                   ______________________________________                                    

The modification method of the present invention described above is a method using carbon dioxide, so that it has high operational safety, requires no high-temperature heating and is capable of producing such effects as an amelioration of smell of liquid foodstuff, a inactivation of enzymes and a sterilization.

Test Example 4

An enzyme-containing test liquid and a carbon dioxide fluid were contacted with each other according to the process illustrated in FIG. 1. As the test liquid, an aqueous solution containing glucoamylase adjusted to a concentration of 20 mg/100 ml, was used. Filters made of a sintered metal and having a mesh size of (i) 2 μm, (ii) 7 μm, (iii) 10 μm, (iv) 50 μm and (v) 100 μm in average diameter were used as a filter 16 provided at the bottom of a treating tank 13. No entrainer was used.

First, 450 ml of the test liquid was supplied into the treating tank 13 (500 ml) while starting the feed of carbon dioxide from a liquefied carbon dioxide gas cylinder 1. Carbon dioxide was passed through a valve 9 and a filter 5, was liquefied by a cooler 6, was heated to 50° C. by a heater 7 and was supplied into the treating tank 13 through a check valve 10, a line mixer 8, a valve 12 and the filter 16 by a pump 3. The pressure and temperature in the treating tank 13 were maintained at 25 MPa and 35° C., respectively, by a tank heater 14 and a pressure regulator 15. The feeding rate of the carbon dioxide fluid into the treating tank 13 was 10 NL/min, and the contact time between enzyme-containing test liquid and the carbon dioxide fluid was 30 minutes. The carbon dioxide (CO₂) concentration in test liquid in the treating tank was 0.65 mol/liter (mesh size: 2 μm), 0.76 mol/liter (mesh size: 7 μm), 0.86 mol/liter (mesh size: 10 μm), 0.70 mol/liter (mesh size: 50 μm) and 0.60 mol/liter (mesh size: 100 μm), respectively. Then, the enzyme-containing test liquid and the carbon dioxide fluid were led into a separating tank 17, and the temperature and pressure of the carbon dioxide fluid in the treating tank 17 were returned to the ordinary level by operating a tank heater 18 and a pressure regulator 19 to release the supercritical state. The gasified carbon dioxide was discharged out of the system through a flowmeter 20.

                  TABLE 8                                                          ______________________________________                                                  Mesh      Mesh   Mesh   Mesh  Mesh                                             size:     size:  size:  size: size:                                   Condition                                                                               2 μm   7 μm                                                                               10 μm                                                                              50 μm                                                                             100 μm                               ______________________________________                                         Residual 2         1      0      4     13                                      enzyme                                                                         activity                                                                       (%)                                                                            ______________________________________                                    

Test Example 5

A test liquid containing microorganisms shown in Table 9 below and a carbon dioxide fluid were contacted with each other according to the process illustrated in FIG. 1. As the test liquid, a physiological salt solution containing the microorganisms adjusted to a concentration of about 10⁶ count/ml, was used. A filter made of a sintered metal and having a mesh size of 10 μm in average diameter was used as a filter 16 provided at the bottom of a treating tank 13. No entrainer was used.

First, 450 ml of the test liquid was supplied into the treating tank 13 (500 ml) while starting the feed of carbon dioxide from a liquefied carbon dioxide gas cylinder 1. Carbon dioxide was passed through a valve 9 and a filter 5, was liquefied by a cooler 6, was heated to 35° C. by a heater 7 and was supplied into the treating tank 13 through a check valve 10, a line mixer 8, a valve 12 and the filter 16 by a pump 3. The pressure and temperature in the treating tank 13 were maintained at 25 MPa and 35° C., respectively, by a tank heater 14 and a pressure regulator 15. The feeding rate of the carbon dioxide fluid into the treating tank 13 was 10 NL/min, and the contact time between microorganism-containing test liquid and the carbon dioxide fluid was 30 minutes (Lactobacillus brevis), or 30 minutes (Saccharomyces cerevisiase), respectively. As shown in Table 9, the excellent effect thereof was obtained in case where the carbon dioxide (CO₂) density of the test liquid in the treating tank was not less than 0.76 g/cm³ (Lactobacillus brevis), or was not less than 0.71 g/cm³ (Saccharomyces cerevisiase), respectively.

Then, the liquid and the carbon dioxide fluid were led into a separating tank 17, and the temperature and pressure of the carbon dioxide fluid in the treating tank 17 were returned to the ordinary level by operating a tank heater 18 and a pressure regulator 19 to release the supercritical state. The gasified carbon dioxide was discharged out of the system through a flowmeter 20.

                  TABLE 9                                                          ______________________________________                                                            Survival                                                    Microorganisms     (count/ml)                                                  ______________________________________                                         Lactobacillus brevis                                                                              0                                                           Saccharomyces cerevisiase                                                                         0                                                           ______________________________________                                    

Test Example 6

A test liquid containing spores shown in Table 10 and a carbon dioxide fluid were contacted with each other according to the process illustrated in FIG. 1. As the test liquid, an acetic acid buffer containing the spores adjusted to a concentration of about 10⁶ (count/ml), was used. A filter made of a sintered metal and having a mesh size of 10 μm in average diameter was used as a filter 16 provided at the bottom of a treating tank 13. No entrainer was used.

First, 450 ml of the test liquid was supplied into the treating tank 13 (500 ml) while starting the feed of carbon dioxide from a liquefied carbon dioxide gas cylinder 1. Carbon dioxide was passed through a valve 9 and a filter 5, was liquefied by a cooler 6, was heated to 40° C. by a heater 7 and was supplied into the treating tank 13 through a check valve 10, a line mixer 8, a valve 12 and the filter 16 by a pump 3. The pressure and temperature in the treating tank 13 were maintained at 30 MPa and 50° C., respectively, by a tank heater 14 and a pressure regulator 15. The feeding rate of the carbon dioxide fluid into the treating tank 13 was 10 NL/min, and the contact time between spores-containing test liquid and the carbon dioxide fluid was 80 minutes. As shown in Table 10, the excellent inactivating effect thereof was obtained respectively.

Then, the liquid and the carbon dioxide fluid were led into a separating tank 17, and the temperature and pressure of the carbon dioxide fluid in the treating tank 17 were returned to the ordinary level by operating a tank heater 18 and a pressure regulator 19 to release the supercritical state. The gasified carbon dioxide was discharged out of the system through a flowmeter 20.

                  TABLE 10                                                         ______________________________________                                                             Survival                                                   Spores              (count/ml)                                                 ______________________________________                                         spores of Bacillus cereus                                                                          0                                                          spores of Bacillus subtilis                                                                        0                                                          spores of Bacillus megaterium                                                                      0                                                          spores of Bacillus polymyxa                                                                        0                                                          spores of Bacillus coagulans                                                                       0                                                          scores of Bacillus circulans                                                                       0                                                          spores of Bacillus  0                                                          licheniformis                                                                  spores of Bacillus macerans                                                                        0                                                          ______________________________________                                    

Test Example 7

A test liquid containing phage virus shown in Table 11 and a carbon dioxide fluid were contacted with each other according to the process illustrated in FIG. 1. As the test liquid, an aqueous solution containing the virus adjusted to a concentration of about 10⁵ (count/ml), was used. A filter made of a sintered metal and having a mesh size of 10 μm in average diameter was used as a filter 16 provided at the bottom of a treating tank 13. No entrainer was used.

First, 450 ml of the test liquid was supplied into the treating tank 13 (500 ml) while starting the feed of carbon dioxide from a liquefied carbon dioxide gas cylinder 1. Carbon dioxide was passed through a valve 9 and a filter 5, was liquefied by a cooler 6, was heated to 40° C. by a heater 7 and was supplied into the treating tank 13 through a check valve 10, a line mixer 8, a valve 12 and the filter 16 by a pump 3. The pressure and temperature in the treating tank 13 were maintained at 30 MPa and 50° C., respectively, by a tank heater 14 and a pressure regulator 15. The feeding rate of the carbon dioxide fluid into the treating tank 13 was 10 NL/min, and the contact time between phage virus-containing test liquid and the carbon dioxide fluid was 30 minutes. As shown in Table 11, the excellent survival effect thereof was obtained, respectively.

Then, the liquid and the carbon dioxide fluid were led into a separating tank 17, and the temperature and pressure of the carbon dioxide fluid in the treating tank 17 were returned to the ordinary level by operating a tank heater 18 and a pressure regulator 19 to release the supercritical state. The gasified carbon dioxide was discharged out of the system through a flowmeter 20.

                  TABLE 11                                                         ______________________________________                                                            Survival                                                    Phage virus        (count/ml)                                                  ______________________________________                                         Escherichia coli T1 phage                                                                         0                                                           Escherichia coli T2 phage                                                                         0                                                           Escherichia coli T3 phage                                                                         0                                                           Escherichia coli T4 phage                                                                         0                                                           Escherichia coli T5 phage                                                                         0                                                           ______________________________________                                    

Test Example 8

A test liquid containing enzymes shown in Table 12 and a carbon dioxide fluid were contacted with each other according to the process illustrated in FIG. 1. As the test liquid, an acetic acid buffer containing the enzymes adjusted to a concentration of 20 mg/100 ml, was used. A filter made of a sintered metal and having a mesh size of 10 μm in average diameter was used as a filter 16 provided at the bottom of a treating tank 13. No entrainer was used.

First, 450 ml of the test liquid was supplied into the treating tank 13 (500 ml) while starting the feed of carbon dioxide from a liquefied carbon dioxide gas cylinder 1. Carbon dioxide was passed through a valve 9 and a filter 5, was liquefied by a cooler 6, was heated to 35° C. by a heater 7 and was supplied into the treating tank 13 through a check valve 10, a line mixer 8, a valve 12 and the filter 16 by a pump 3. The pressure and temperature in the treating tank 13 were maintained at 25 MPa and 35° C., respectively, by a tank heater 14 and a pressure regulator 15. The feeding rate of the carbon dioxide fluid into the treating tank 13 was 10 NL/min, and the contact time between enzyme-containing test liquid and the carbon dioxide fluid was 30 minutes. As shown in Table 12, the excellent effect thereof was obtained, respectively.

Then, the liquid and the carbon dioxide fluid were led into a separating tank 17, and the temperature and pressure of the carbon dioxide fluid in the treating tank 17 were returned to the ordinary level by operating a tank heater 18 and a pressure regulator 19 to release the supercritical state. The gasified carbon dioxide was discharged out of the system through a flowmeter 20.

                  TABLE 12                                                         ______________________________________                                                        Residual                                                                       enzyme activity                                                                (%)                                                             ______________________________________                                         Glucoamylase     17                                                            Acid protease    11                                                            Pectinase        2                                                             Carboxy peptidase                                                                               0                                                             Lipase           0                                                             α-Amylase  4                                                             β-Amylase   2                                                             Catalase         0                                                             Lipoxigenase     0                                                             ______________________________________                                    

Test Example 9

A test liquid containing microorganisms shown in Table 13 and a carbon dioxide fluid were contacted with each other according to the process illustrated in FIG. 1. As the test liquid, a physiological salt solution containing the microorganisms adjusted to a concentration of about 10⁶ count/ml, was used. A filter made of a sintered metal and having a mesh size of 10 μm in average diameter was used as a filter 16 provided at the bottom of a treating tank 13. No entrainer was used.

First, 450 ml of the test liquid was supplied into the treating tank 13 (500 ml) while starting the feed of carbon dioxide from a liquefied carbon dioxide gas cylinder 1. Carbon dioxide was passed through a valve 9 and a filter 5, was liquefied by a cooler 6, was heated to 35° C. by a heater 7 and was supplied into the treating tank 13 through a check valve 10, a line mixer 8, a valve 12 and the filter 16 by a pump 3. The pressure and temperature in the treating tank 13 were maintained at 25 MPa and 35° C., respectively, by a tank heater 14 and a pressure regulator 15. The feeding rate of the carbon dioxide fluid into the treating tank 13 was 10 NL/min, and the contact time between microorganism-containing test liquid and the carbon dioxide fluid was 30 minutes. As shown in Table, the excellent effect thereof was obtained, respectively.

Then, the liquid and the carbon dioxide fluid were led into a separating tank 17, and the temperature and pressure of the carbon dioxide fluid in the treating tank 17 were returned to the ordinary level by operating a tank heater 18 and a pressure regulator 19 to release the supercritical state. The gasified carbon dioxide was discharged out of the system through a flowmeter 20.

                  TABLE 13                                                         ______________________________________                                                           Survival                                                     Microorganisms    (count/ml)                                                   ______________________________________                                         Escherichia coli  0                                                            Bacillus cereus   0                                                            Bacillus subtilis 0                                                            Bacillus magaterium                                                                              0                                                            Bacillus polymyxa 0                                                            Penicillium frequentans                                                                          0                                                            Penicillium jenseni                                                                              0                                                            Aspergillus oryzae                                                                               0                                                            Sacchromyces cerevisiae                                                                          0                                                            ______________________________________                                     

What is claimed is:
 1. A method for inactivating enzymes, microorganisms and spores in a liquid foodstuff comprising contacting the liquid foodstuff in a treating tank with carbon dioxide in a supercritical state supplied through a filter with a mesh size of not more than 120 μm average diameter at a carbon dioxide concentration in the liquid foodstuff of not less than 0.4 mole/liter for 10 seconds to 120 minutes, thereby inactivating enzymes, microorganisms and spores present in the liquid foodstuff wherein the carbon dioxide density in the liquid foodstuff is not less than 0.4 g/cm³, and the pressure in the treating tank is not less than 15 MPa.
 2. The method according to claim 1, wherein the liquid foodstuff is a fermented liquid foodstuff.
 3. The method according to claim 1, wherein the liquid foodstuff is a fruit juice.
 4. The method according to claim 1, wherein the mesh size of the filter is 1 to 100 μm in average diameter.
 5. The method according to claim 1, wherein the temperature of the liquid foodstuff in the treating tank is 30° to 80° C.
 6. A method for sterilizing microorganisms, spores and virus in a liquid foodstuff comprising contacting the liquid foodstuff in a treating tank with carbon dioxide in a supercritical state supplied through a filter with a mesh size of not more than 120 μm in average diameter at a carbon dioxide concentration in the liquid foodstuff of not less than 0.4 mole/liter for 10 seconds to 120 minutes, thereby sterilizing microorganisms, spores and virus present in the liquid foodstuff wherein the carbon dioxide density in the liquid foodstuff is not less than 0.4 g/cm³, and the pressure in the treating tank is not less than 15 MPa.
 7. The method according to claim 6, wherein the liquid foodstuff is a fermented liquid foodstuff.
 8. The method according to claim 6, wherein the liquid foodstuff is a fruit juice.
 9. The method according to claim 6, wherein the mesh size of the filter is 1 to 100 μm in average diameter.
 10. The method according to claim 6, wherein the temperature of the liquid foodstuff in the treating tank is 30° to 80° C. 